Two of the four sections of this proposal deal with synthesis of N-linked glycoproteins via the dolichol mediated pathway. The first section is concerned with synthesis of complex and polymannose chains of N-linked proteins. To investigate the mechanism that determines the type of chain synthesized on a polypeptide we will utilize our finding that unfolded proteins containing the sequence -Asn-X-Ser/Thr- can be glycosylated in vitro. Since porcine RNase is known to contain complex chains at Asn21 and Asn76 and a polymannose chain at Asn34, we will utilize the unfolded, unglycosylated form of this protein to study the in vitro introduction and processing to these three oligosaccharide chains. The second section concerns the metabolism of dolichol (Dol) and Dol esters. Using oviduct tissue slices and cell free preparations we will determine if Dol kinase, Dol ester esterase, Dol phosphate phosphatase and Dol pyrophosphate pyrophosphatase are present. To define the metabolism of these four Dol compounds in relation to the role of Dol phosphate as a carrier in glycoprotein synthesis, their interconversion will be studied in oviduct tissue slices that synthesize ovalbumin. Interconversions will be studied under control conditions, and conditions in which either glycosylation or Dol synthesis is blocked. The third section deals with synthesis of O-linked glycoproteins. A system for in vitro translation of submaxillary apomucin will be devised. The subcellular sites of this synthesis and glycosylation in submaxillary gland will be established. Studies to define the mechanism of its glycosylation (presumably occurring in the Golgi complex) will be undertaken. Lastly, we will investigate the possibility that one or more subunits of the mitochondrial F1-ATPase is a glycoprotein, since this enzyme has been reported to contain carbohydrate. If it does, we will structurally characterize the oligosaccharide chain. Then, by the use of various glycosidases, we will study the role of the oligosaccharide chain in the following properties of the F1-ATPase: its ATP hydrolytic activity; the interaction of its five subunits; and its ability to interact with the inner mitochondrial membrane to form a fully functional F0f1-ATPase.